The polyamines, putrescine, spermidine, and spermine, are major polybasic compounds in all living cells. These amines are important for many systems related to growth and differentiation. For many years we have been studying how these polyamines are synthesized, how their biosynthesis and degradation are regulated, their physiologic functions, how they act in vivo, and the structure of the various biosynthetic enzymes. For this purpose we have constructed null mutants in each of the biosynthetic steps in both Escherichia coli and Saccharomyces cerevisiae, and have prepared overexpression systems for the biosynthetic enzymes. Our overall studies have aimed at the use of these mutants to elucidate the physiological functions of the polyamines, and, in particular, to ascertain the physiological effects of polyamine deprivation. During the past year we have concluded our studies on the involvement of methylthioadenosine in the polyamine biosynthetic pathway, and have shown that this pathway accounts for greater than 98% of the methylthioadenosine formed by the cell. In addition we have shown that the polyamine biosynthetic pathway accounts for about 15% of the metabolism of methionine. In other studies we have shown that polyamine-deficient S. cerevisiae cells accumulate reactive oxygen species and develop an apoptotic phenotype. These effects can be prevented by very low concentrations of spermidine, and we have shown that the internal concentrations of spermidine required are far less than the internal concentration of spermidine normally found in S. cerevisiae cells. Our current studies have concentrated on the use of microarray and proteomic techniques to study the changes caused by various concentrations of spermidine. Since changes in growth rate would complicate the effects observed after addition of spermidine, we have standardized a chemostat procedure for use in these studies.